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Biomedical subjects

D R VanDevanter

Publications and source records attributed to D R VanDevanter.

14 recordsLinked to original sources
PubMed2002

The Pseudomonas aeruginosa genome: how do we use it to develop strategies for the treatment of patients with cystic fibrosis and Pseudomonas infections?

In the 2 years since the complete sequence of Pseudomonas aeruginosa strain PAO1 was published, at least 200 papers have been published describing research that made use of the PAO1 genome sequence. Some of this research included genome-wide studies of gene expression or the effect of mutation on bacterial functions such as biofilm formation; this type of global analysis would not have been possible without the availability of the sequence. As a result of these and other, more traditional, research studies, there is a wealth of new knowledge about the physiology of this pathogen. This raises the possibility of new strategies for the treatment of patients with P. aeruginosa infection, either by novel antibiotics or by drugs targeting bacterial functions essential for survival and virulence in the human host.

Biofilms↗
PubMed2000

A small-molecule nitroimidazopyran drug candidate for the treatment of tuberculosis.

Mycobacterium tuberculosis, which causes tuberculosis, is the greatest single infectious cause of mortality worldwide, killing roughly two million people annually. Estimates indicate that one-third of the world population is infected with latent M. tuberculosis. The synergy between tuberculosis and the AIDS epidemic, and the surge of multidrug-resistant clinical isolates of M. tuberculosis have reaffirmed tuberculosis as a primary public health threat. However, new antitubercular drugs with new mechanisms of action have not been developed in over thirty years. Here we report a series of compounds containing a nitroimidazopyran nucleus that possess antitubercular activity. After activation by a mechanism dependent on M. tuberculosis F420 cofactor, nitroimidazopyrans inhibited the synthesis of protein and cell wall lipid. In contrast to current antitubercular drugs, nitroimidazopyrans exhibited bactericidal activity against both replicating and static M. tuberculosis. Lead compound PA-824 showed potent bactericidal activity against multidrugresistant M. tuberculosis and promising oral activity in animal infection models. We conclude that nitroimidazopyrans offer the practical qualities of a small molecule with the potential for the treatment of tuberculosis.

Animals↗
PubMed1996

Unusual expression of mRNA typical of Philadelphia positive acute lymphoblastic leukemia detected in chronic myeloid leukemia.

The Philadelphia chromosome (Ph) is found in both chronic myeloid leukemia (CML) and acute lymphoblastic leukemia (ALL). The Ph translocation, t(9;22)(q34;q11), can disrupt the BCR gene on chromosome 22 in one to two areas called the major (Mbcr1) and minor (mbcr1) breakpoint cluster regions. In CML the breakpoint has been mapped almost exclusively to Mbcr1, whereas in Ph positive ALL both Mbcr1 and the upstream mbcr1 breakpoints have been described. In this communication we describe an unusual patient with typical chronic phase Ph positive CML and evidence of the uncharacteristic mbcr1 breakpoint, predicting expression of the ALL-type p190 fusion protein. Fluorescence in situ hybridization demonstrated BCR gene rearrangement, the reverse transcription polymerase chain reaction detected the BCR-ABL fusion mRNA characteristic of the mbcr1 breakpoint, and failed to detect BCR-ABL mRNA characteristic of the Mbcr1 breakpoint. Southern blot analysis revealed no rearrangement in Mbcr1, and direct sequencing of the PCR product confirmed it to be the ALL-type mbcr1 fusion mRNA with the first exon of the BCR gene fused to ABL exon a2. This case differs from the previously reported cases of "p190" CML in that the patient presented without abnormal hematopoietic features other than those found in typical CML and provides further evidence that the p190 mRNA is not sufficient to cause an acute rather than chronic leukemia.

Base Sequence↗
PubMed1996

Detection and analysis of diverse herpesviral species by consensus primer PCR.

A consensus primer PCR method which amplifies a region of herpesviral DNA-directed DNA polymerase (EC 2.7.7.7) and which uses degenerate primers in a nested format was developed. Primers were designed to target sequences coding for highly conserved amino acid motifs covering a region of approximately 800 bp. The assay was applied to 22 species of herpesviruses (8 human and 14 animal viruses), with PCR products obtained for 21 of 22 viruses. In the process, 14 previously unreported amino acid-coding sequences from herpesviral DNA polymerases were obtained, including regions of human herpesviruses 7 and 8. The 50 to 60 amino acid-coding sequences recovered in the present study were determined to be unique to each viral species studied, with very little sequence variation between strains of a single species when studied. Template dilution studies in the presence of human carrier DNA demonstrated that six human herpesviruses (herpesviruses 1, 2, 3, 4, 5, and 6B) could be detected at levels at or below 100 genome equivalents per 100 ng of carrier DNA. These data suggest that consensus primer PCR targeted to herpesviral DNA polymerase may prove to be useful in the detection and identification of known herpesviruses in clinical samples and the initial characterization of new herpesviral genomes.

Amino Acid Sequence↗
PubMed1995

Electrophoretic isolation of extrachromosomal DNA from tumor cells.

Gene amplification allows transformed cells to overexpress specific genes and gain a survival advantage. For this reason, cloning and characterization of amplified genes can improve our understanding of the biology of transformed cells. The techniques of in-gel renaturation and chromosome microdissection can enrich for amplified DNA sequences, but both are labor intensive and have other drawbacks. We have developed an alternative strategy of enriching for amplified DNA sequences that involves two-directional agarose gel electrophoresis of extrachromosomal circular DNA. Extrachromosomal circles can be detected with repetitive DNA probes and can be used to produce DNA probes suitable for fluorescence in situ hybridization for location of genomic origin. The ability to enrich for amplified DNA without specialized equipment or transformed cell metaphases should prove useful in the search for new genes which are important in tumor cell progression.

Base Sequence↗
PubMed1994

Cloning of the LamA3 gene encoding the alpha 3 chain of the adhesive ligand epiligrin. Expression in wound repair.

We have isolated cDNA clones encoding the entire 170-kDa chain of epiligrin (alpha 3Ep) and a genomic clone encoding the alpha 3Ep gene (LamA3). Analysis of multiple cDNA clones revealed two distinct transcripts (alpha 3EpA and alpha 3EpB). Sequencing of the alpha 3EpA transcript indicated sequence and structural homology to laminin alpha 1 and alpha 2 chains that extend from domain IIIa through the carboxyl-terminal G domain. The alpha 3EpB transcript encodes a larger amino-terminal domain and contains additional epidermal growth factor repeats and sequences corresponding to domain IV of alpha 1 laminin. Fluorescence in situ hybridization indicated that the LamA3 gene is located on chromosome 18q11.2, a locus distinct from the LamA1 gene (18p11.3). The G domain of the epiligrin alpha 3 chain contains five subdomains that are individually related to the G subdomains reported for Drosophila and vertebrate laminin alpha chains. Sequence divergence within the G domain of alpha 3 epiligrin suggests that it is functionally distinct from laminin, consistent with our previous report showing that epiligrin interacts with different integrin adhesion receptors. Analysis of RNA from human foreskin keratinocytes (HFKs) identified multiple epiligrin transcripts that were down-regulated by viral transformation and differentiation. In contrast, epiligrin expression was up-regulated in wound sites of human skin.

Amino Acid Sequence↗
PubMed1994

Pure chromosome-specific PCR libraries from single sorted chromosomes.

Chromosome-specific DNA libraries can be very useful in molecular and cytogenetic genome mapping studies. We have developed a rapid and simple method for the generation of chromosome-specific DNA sequences that relies on polymerase chain reaction (PCR) amplification of a single flow-sorted chromosome or chromosome fragment. Previously reported methods for the development of chromosome libraries require larger numbers of chromosomes, with preparation of pure chromosomes sorted by flow cytometry, generation of somatic cell hybrids containing targeted chromosomes, or a combination of both procedures. These procedures are labor intensive, especially when hybrid cell lines are not already available, and this has limited the generation of chromosome-specific DNA libraries from nonhuman species. In contrast, a single sorted chromosome is a pure source of DNA for library production even when flow cytometric resolution of chromosome populations is poor. Furthermore, any sorting cytometer may be used with this technique. Using this approach, we demonstrate the generation of PCR libraries suitable for both molecular and fluorescence in situ hybridization studies from individual baboon and canine chromosomes, separate human homologues, and a rearranged marker chromosome from a transformed cell line. PCR libraries specific to subchromosomal regions have also been produced by sorting a small chromosome fragment. This simple and rapid technique will allow generation of nonhuman linkage maps and probes for fluorescence in situ hybridization and the characterization of marker chromosomes from solid tumors. In addition, allele-specific libraries generated by this strategy may also be useful for mapping genetic diseases.

Animals↗
PubMed1994

Y chromosome loss in chronic myeloid leukemia detected in both normal and malignant cells by interphase fluorescence in situ hybridization.

Loss of the Y chromosome in bone marrow (BM) cells is a normal age-associated event. Y chromosome loss is also observed in the Philadelphia chromosome (Ph) positive BM cells of some patients with chronic myeloid leukemia (CML) in chronic phase, but at a younger age than in normal individuals. While the significance of loss of the sex chromosome in normal males is uncertain, -Y marrow cells are not believed to be of clonal origin. However, because CML is a clonal disease, CML sub-populations with Y loss may constitute a disease-related sub-clone. We used a PCR-amplified yeast artificial chromosome containing the BCR gene region for single color interphase analysis of BCR rearrangement by fluorescence in situ hybridization (FISH). Then, using two color FISH, with one fluorochrome detecting the BCR gene region and the other detecting Y chromosome repeat sequences, we surveyed peripheral and BM Y loss in both normal Ph- (BCR not disrupted) and CML Ph+ (BCR rearranged) interphase nuclei of two patients with Y loss in Ph positive cells observed by metaphase analysis. -Y was seen in a proportion of Ph+ cells in both cases, and the proportion matched that seen in Ph- cells, indicating that Y loss is probably sporadic in both normal and CML populations, and that the propensity for Y loss in normal BM cells may be a phenotype that can be retained by malignant cells in CML.

Chromosome Deletion↗
PubMed1993

Recombination between separate MYC amplification structures in COLO320 cells.

Cytogenetically visible gene amplification structures can consist of arrays of amplicons presumably formed by secondary "rearrangements" following amplicon formation. The structural evolution of gene amplification sites in tumor cells suggests that complex secondary structures may have some selective advantage in the tumor cell environment. Although secondary amplicon rearrangements are a hallmark of the gene amplification process, little is known about the mechanics of this process. COLO320 neuroendocrine tumor cells carry two different types of amplified MYC oncogene sequences, one type with an intact MYC gene and the other with a rearranged "chimeric" MYC gene. We have studied various clonal subpopulations of COLO320 cells and identified regions within and downstream of the MYC locus that are unique to each amplicon type. Using double-label fluorescence in situ hybridization with DNA probes unique to each amplicon type, we have observed that both chromosomal and extrachromosomal MYC amplicon arrays in COLO320 cells frequently consist of heterogeneous mixtures of each MYC amplicon type. Our results suggest that the two MYC amplicon types of COLO320 cells were formed simultaneously but independently, and that double minute chromosomes observed in COLO320 cells were formed by intermolecular homologous recombination secondary to amplicon formation.

Adenocarcinoma↗
PubMed1992

Preparation of uniform, intact DNA samples from resected tumor tissues for pulsed-field gel electrophoretic analyses.

Preparation of high molecular weight DNA from resected tumor tissues suitable for pulsed-field gel electrophoresis (PFGE) can be complicated by the presence of nonviable cells and lymphocytes. We have developed a simple procedure to reduce the level of degraded DNA in PFGE DNA samples prepared from resected tumor tissues. The procedure employs a single, three component Percoll step gradient centrifugation and can be performed on several tumor samples simultaneously. Analyses of DNAs from 15 tumor specimens (7 solid tumors and 8 aspirated fluids) demonstrate that the technique enriches the integrity of PFGE DNA samples. Morphologic evaluation of 9 specimens suggested that both cellular debris and contaminating normal lymphocytes are removed from starting cell populations during the enrichment procedure. Fractionation of cells also reduced cell clumping, allowing for the formation of more uniform PFGE DNA samples.

Centrifugation, Density Gradient↗
PubMed1991

Trisomy 8 in primary esthesioneuroblastoma.

Esthesioneuroblastoma is a rare malignancy believed to be derived from neuroectodermal stem cells within the olfactory epithelium. We have obtained the karyotype of a primary esthesioneuroblastoma following brief (7-day) in vitro culture, and have determined that the only observable cytogenetic anomaly is the presence of an additional chromosome 8. Previously, the karyotypes of two cell lines established from metastatic esthesioneuroblastomas have been reported to contain the equivalent of three copies of chromosome 8, in addition to other chromosomal aberrations, including the reciprocal translocation, t(11;22)(q24;q12). Examination of the cytogenetic literature suggests that an extra copy of chromosome 8 is a common occurrence in undifferentiated small round cell tumors frequently observed to carry the t(11;22), including esthesioneuroblastoma, Ewing's sarcoma, peripheral neuroepithelioma, Askin's tumor, and rhabdomyosarcoma. These data, combined with our report of a small round cell tumor with the karyotype 47,XY, +8, indicate that trisomy 8 may be a common phenomenon in these tumors, and may also provide some sort of selective advantage to these tumor types.

Chromosome Aberrations↗
PubMed1990

Ability of circular extrachromosomal DNA molecules to carry amplified MYCN proto-oncogenes in human neuroblastomas in vivo.

Amplification of the proto-oncogene MYCN (also known as N-myc) in neuroblastomas has been shown to correlate with both disease stage and prognosis, yet little is known about the DNA structures that carry amplified MYCN genes in neuroblastomas in vivo. We have used DNA irradiation and pulsed-field gel electrophoresis to analyze MYCN amplification structures in eight neuroblastomas from separate patients (four primary tumors and four metastatic lesions exhibiting MYCN amplification). Six of the eight neuroblastomas (three primary tumors and three metastatic lesions) exhibited MYCN DNA irradiation profiles consistent with the presence of circular extrachromosomal DNA amplification structures. Five neuroblastomas possessed amplification structures within the size range of double minute chromosomes, and one contained smaller DNA circles. Two neuroblastomas exhibited MYCN DNA irradiation patterns consistent with larger (presumably chromosomal) amplification structures. Multiple sizes of DNA circles were observed in the neuroblastomas of four different patients, implying in vivo multimerization of amplification structures. The presence of circular MYCN amplification structures in six of eight neuroblastomas examined suggests that circular DNA molecules are important structures in in vivo gene amplification.

Blotting, Southern↗
PubMed1990

Loss of DNA-membrane interactions and cessation of DNA synthesis in myeloperoxidase-treated Escherichia coli.

Neutrophils and monocytes employ a diverse array of antimicrobial effector systems to support their host defense functions. The mechanisms of action of most of these systems are incompletely understood. The present report indicates that microbicidal activity by a neutrophil-derived antimicrobial system, consisting of myeloperoxidase, enzymatically generated hydrogen peroxide, and chloride ion, is accompanied by prompt cessation of DNA synthesis in Escherichia coli, as determined by markedly reduced incorporation of [3H]thymidine into trichloracetic acid-precipitable material. Simultaneously, the myeloperoxidase system mediates a decline in the ability of E. coli membranes to bind hemimethylated DNA sequences containing the E. coli chromosomal origin of replication (oriC). Binding of oriC to the E. coli membrane is an essential element of orderly chromosomal DNA replication. Comparable early changes in DNA synthesis and DNA-membrane interactions were not observed with alternative oxidant or antibiotic-mediated microbicidal systems. It is proposed that oxidants generated by the myeloperoxidase system modify the E. coli membrane in such a fashion that oriC binding is markedly impaired. As a consequence chromosomal DNA replication is impaired and organisms can no longer replicate.

Cell Membrane↗